Non-aggregating non-quenching oligomer comprising nucelotide analogs, method of synthesis and use thereof

ABSTRACT

The invention provides compositions and methods for improved hybridization analysis utilizing DNA, RNA, PNA and chimeric oligomers in which one or more purine bases are substituted by a pyrazolo[5,4-d]pyrimidine or by a 7-deazapurine purine analogue. Reduced self-aggregation and reduced fluorescence quenching are obtained when the oligomers are used in various methods involving hybridization. Methods of synthesis, as well as novel synthetic precursors, are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of application Ser. No. 09/447,936filed Nov. 23, 1999 now U.S. Pat. No. 6,660,845.

TECHNICAL FIELD

The disclosure concerns the use of nucleotide analogues to provideimproved properties to hybridization probes, including DNA and RNAprobes and modified nucleic acid probes, such as peptide nucleic acids(PNAs), and to chimeric probes containing two or more types of nucleicacid and/or modified nucleic acid.

BACKGROUND

Hybridization analysis is central to a variety of techniques inmolecular biology and diagnostics, including gene cloning, geneidentification, forensic analysis, pharmacogenomics and identificationof genetic polymorphisms. Hybridization can be used as an endpoint of anassay, whereby the presence of hybridized probe constitutes the readoutfor the assay; or hybridization can be used as an initial step in anassay, wherein an event subsequent to hybridization (such as, forexample, extension of a hybridized primer or hydrolysis of a hybridizedprobe) is used as the readout.

Traditionally, hybridization probes and primers have been DNA molecules;however, there are certain disadvantages to the use of DNA as a probe orprimer. For example, the base composition of a DNA molecule can affectits effectiveness as a probe or primer in several ways. A DNA moleculewith a high concentration of G residues is often difficult to handle(e.g., problems with aggregation and poor solubility) and can yield highbackground in hybridization reactions. It is also well-known that G-richDNA molecules are prone to the production of artifacts in the analysisof DNA sequences by gel electrophoresis, presumably due to the adoptionof secondary structure by such molecules, despite the denaturingconditions under which such analyses are conducted.

Various modified forms of DNA and DNA analogues have been used inattempts to overcome some of the disadvantages of the use of DNAmolecules as probes and primers. Among these are peptide nucleic acids(PNAs, also known as polyamide nucleic acids). Nielsen et al. (1991)Science 254:1497-1500. PNAs contain heterocyclic base units, as found inDNA and RNA, that are linked by a polyamide backbone, instead of thesugar-phosphate backbone characteristic of DNA and RNA. PNAs are capableof hybridization to complementary DNA and RNA target sequences and, infact, hybridize more strongly than a corresponding nucleic acid probe.Furthermore, PNAs are resistant to many types of nuclease which attackthe sugar-phosphate DNA and RNA backbones. Additional advantages of PNAsinclude the ability of specifically modified PNAs to cross theblood-brain-barrier and the observation that PNAs injectedintrathecally, can mediate antisense affects in vivo. During et al.(1999) Nature Biotechnol. 17:753-754.

The synthesis of PNA oligomers and reactive monomers used in thesynthesis of PNA oligomers have been described in U.S. Pat. Nos.5,539,082; 5,714,331; 5,773,571; 5,736,336 and 5,766,855. Alternateapproaches to PNA synthesis and monomers for PNA synthesis have beensummarized. Uhlmann et al. (1998) Angew. Chem. Int. Ed. 37:2796-2823.

However, as they become more widely used, disadvantages of PNAs are alsobecoming apparent. For example, long PNA oligomers, depending on theirsequence, are prone to aggregation, difficult to purify and difficult tocharacterize. In addition, purine-rich PNA oligomers tend to aggregateand are poorly soluble in aqueous media. Gangamani et al. (1997)Biochem. Biophys. Res. Comm. 240:778-782; Egholm, Cambridge HealthtechInstitute 's Seventh Annual Nucleic Acid-Based Technologies, Jun. 21-23,1999, Washington, D.C.; Uhlmann, Cambridge Healthtech Institute'sSeventh Annual Nucleic Acid-Based Technologies, Jun. 21-23, 1999,Washington, D.C. Consequently, effective use of PNAs in hybridization islimited to sequences in which there are no more than 4-5 consecutivepurines, no more than 6 purines in any 10-base portion of the sequence,and/or no more than 3 consecutive G residues. See, for example,

http://www.resgen.com/perseptivedesign.html. Furthermore, since PNA-PNAinteractions are even stronger than PNA-DNA interactions, PNA-containingprobes and primers containing self-complementary sequences cannotgenerally be used for hybridization to a target sequence. Anotherconsequence of the very strong interaction between PNAs andcomplementary DNA and/or RNA molecules is that it is difficult to obtainsingle nucleotide mismatch discrimination using PNA probes. Demidov etal. (1995) Proc. Natl. Acad. Sci. USA 92:2637-2641

Uhlmann et al., supra reviewed approaches for increasing the solubilityof PNAs, including synthesis of PNA/DNA chimeras and addition ofterminal lysine residues to a PNA oligomer. They did not disclose theuse of nucleotide analogues to increase solubility and improvehybridization properties of PNA oligomers.

Similar design constraints are required in the synthesis ofnon-PNA-containing oligonucleotide probes and primers. See, for example,the publication entitled “Sequence Detection Systems Quantitative AssayDesign and Optimization,” PE Biosystems, Stock No. 117 MI02-01. In thesecases, the G/C content of an oligomer must be kept within the range of20-80% and runs of an identical nucleotide, particularly guanine (G),should be avoided. In particular, the aforementioned publication advisesagainst stretches of four or more G residues and against the presence ofa G residue at the 5′ end of a 5′-fluorescently labeled probe. In thecase of primers, the five nucleotides at the 3′ end should comprise nomore than two G and/or C residues.

The synthesis of pyrazolo[3,4-d]pyrimidine and 7-deazapurinenucleosides, as well as their phosphoramidite monomers for use inoligomer synthesis, have been described. Seela et al. (1985) Nucl. AcidsRes. 13:911-926; Seela et al. (1986a) Helv. Chim. Acta 69:1191-1198;Seela et al (1986b) Helv. Chim. Acta 69:1813-1823; and Seela et al.(1987) Biochem. 26:2232-2238. Pyrazolo[3,4-d]pyrimidine and7-deazapurine nucleosides for use in DNA sequencing and as antiviralagents are disclosed in EP 286 028. Co-owned PCT publication WO 99/51775discloses the use of pyrazolo[3,4-d]pyrimidine containingoligonucleotides for hybridization and mismatch discrimination. It hasbeen reported that incorporation of 2′-deoxy-7-deazaguanosine into DNAeliminates band compression in GC-rich stretches during DNA sequenceanalysis by gel electrophoresis (U.S. Pat. No. 5,844,106), decreasestetraplex formation by G-rich sequences (Murchie et al. (1994) EMBO J.13:993-1001) and reduces formation of aggregates characteristic of DNAmolecules containing 2′-deoxyguanosine (U.S. Pat. No. 5,480,980).However, substitution of oligonucleotides with either 7-deazaadenosine(in place of A) or 7-deazaguanosine (in place of G) lowers the T_(m) ofhybrids formed by such substituted oligonucleotides, with greater thanone degree reduction in T_(m) per substituted base. Seela et al. (1987)supra; and Seela et al. (1986) Nucl. Acids Res. 14:2319-2332.

On the other hand, stabilization of duplexes by pyrazolopyrimidine baseanalogues has been reported. Seela et al. (1988) Helv. Chim. Acta.71:1191-1198; Seela et al. (1988) Helv. Chim. Acta. 71:1813-1823; andSeela et al. (1989) Nucleic Acids Res. 17:901-910. Oligonucleotides inwhich one or more purine residues have been substituted bypyrazolo[3,4-d]pyrimidines display enhanced duplex- and triplex-formingability, as disclosed, for example, in Belousov et al. (1998) NucleicAcids Res. 26:1324-1328; U.S. Pat. No. 5,594,121 and co-owned PCTpublication WO 98/49180. Pyrazolo[3,4-d]pyrimidine residues inoligonucleotides are also useful as sites for attachment of variouspendant groups to oligonucleotides. See co-owned PCT Publication WO90/14353, Nov. 29, 1990 and U.S. Pat. No. 5,824,796. None of thesereferences disclose the use of pyrazolopyrimidines or any other type ofbase analogue for reducing aggregation and/or increasing solubility ofan oligomer, or for decreasing quenching of a fluorophore conjugated toan oligomer.

Conjugates comprising a minor groove binder (MGB), an oligonucleotidewherein one or more purine residues are substituted by apyrazolo[3,4-d]pyrimidine (PZP) residue, a fluorophore and afluorescence quencher have been disclosed in co-owned PCT publicationsWO 99/51621 and WO 99/51775. Such conjugates are used, among otherthings, as hybridization probes, primers and hydrolyzable probes in5′-nuclease-based amplification assays. Inclusion of a MGB in theseconjugates increases the stability of hybrids formed by theoligonucleotide portion of the conjugate, allowing the design of shorterprobes. In addition, both the MGB and the PZP contribute to the abilityof such conjugates to exhibit enhanced mismatch discrimination. Neitherof the aforementioned publications disclose the use of PZPs or any othertype of base analogue for reducing aggregation and/or increasingsolubility of an oligomer, or for decreasing quenching of a fluorophoreconjugated to an oligomer.

SUMMARY

Oligomers wherein at least one of the subunits comprises apyrazolopyrimidine and/or a pyrrolopyrimidine base analogue areprovided. The oligomers can comprise DNA, RNA, PNA, or any combinationor chimera thereof. Any number of purine residues in the oligomer can besubstituted by a base analogue. Any of the above-mentioned oligomers cancomprise additional moieties such as fluorophores, fluorescencequenchers and/or minor groove binders.

Oligomers wherein at least one of the subunits comprises apyrazolopyrimidine and/or a pyrrolopyrimidine base analogue, when usedfor hybridization, are less prone to aggregation and self-association,are more soluble, are capable of enhanced mismatch discrimination, anddo not quench the emission of conjugated fluorescent labels.

Oligomers comprising one or more PNA residues wherein at least one ofthe PNA residues comprises a pyrazolopyrimidine and/or apyrrolopyrimidine base analogue are also provided. The oligomers cancomprise exclusively PNA residues, or the oligomers can comprise bothPNA and/or DNA and/or RNA nucleotide residues to constitute a PNA/DNA,PNA/RNA or PNA/DNA/RNA chimera. Any number of purine residues in theoligomer can be substituted by a base analogue. Any of theabove-mentioned oligomers can comprise, additional moieties such asfluorophores, fluorescence quenchers and/or minor groove binders.

In another embodiment, compositions comprising a polymer and afluorophore are provided, wherein one or more purine-containing residuesof the polymer are substituted with a residue comprising apyrazolopyrimidine and/or pyrrolopyrimidine base analogue. Polymers cancomprise PNA, DNA, RNA or any combination or chimera thereof; and thebase analogue can be present in any of the PNA, DNA or RNA portions of achimeric polymer. Any number of purine residues in the polymer can besubstituted by a base analogue, in any of the PNA, DNA and/or RNAportions. The above-mentioned compositions can optionally comprise afluorescence quencher and/or a minor groove binder.

In the polymer-fluorophore compositions just described, quenching of thefluorophore by purine residues in the polymer is reduced when one ormore purines are substituted with a base analogue. Such compositionsadditionally comprising a fluorescence quencher are useful, for example,as probes in hydrolyzable probe assays, in which quenching of thefluorophore by the fluorescence quencher is relieved byhybridization-dependent hydrolysis of probe. The reduction in quenchingafforded by substitution of a base analogue for a purine leads to higherfluorescence output after hydrolysis and, hence, greater sensitivity insuch assays.

New intermediates for the synthesis of PNA-containing oligomerscomprising base analogues are also provided. In one embodiment, aceticacid derivatives of pyrazolopyrimidine and pyrrolopyrimidine baseanalogues, wherein N¹ of the pyrazolopyrimidine or pyrrolopyrimidine islinked to C2 of an acetic acid moiety and functional groups are blocked,are provided. These derivatives are useful for preparation of monomersfor automated synthesis of substituted PNAs and PNA/DNA chimeras.Preferred embodiments of these intermediates include2-{6[(1E)-1-aza-2-(dimethylamino)vinyl]-4-hydroxypyrazolo[5,4-d]pyrimidinyl}aceticacid; 2-(6-amino-4-hydroxypyrazolo[5,4-d]pyrimidinyl) acetic acid; and2-(−4-aminopyrazolo[5,4-d]pyrimidinyl) acetic acid.

Also provided are aminoethylglycyl derivatives of the aforementionedacetic acid derivatives of pyrazolopyrimidine and pyrrolopyrimidine baseanalogues, wherein the α-amino group of a blocked glycyl moiety isderivatized to acetic acid C1 of the acetate and to C2 of an ethylaminemoiety. These derivatives are also known as “PNA monomers.” Suchcompounds are useful for automated synthesis of the aforementionedoligomers and polymers. Preferred embodiments of PPG-containing PNAmonomers (also known as PPPG) include5-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]-3-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-4-oxopentanoicacid and1-{6-[(1E)-aza-2-(dimethylamino)vinyl]-4-hydroxypyrzolo[5,4-d]pyrimidinyl}-N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-N-(2-oxypropyl)acetamide.A preferred embodiment of a PPA-containing PNA monomer is2-[N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}acetylamino]aceticacid.

Also provided are methods for the synthesis of oligomers comprising PNA,DNA, RNA and/or chimeras thereof, wherein the aforementioned PNAmonomers are used at one or more steps in the synthesis. Oligomerssynthesized by these methods are also provided.

In another embodiment, methods for detecting a target sequence in apolynucleotide by hybridization to a probe comprising a DNA, PNA, orPNA/DNA oligomer, wherein one or more residues in the probe comprises apyrazolopyrimidine or pyrrolopyrimidine base analogue, are provided. Inthe practice of these methods, the probe can additionally comprise oneor more of a ribonucleoside, a fluorophore, a fluorescence quencherand/or a minor groove binder.

In another embodiment, methods for detection of a target sequence in apolynucleotide utilizing compositions comprising a polymeric portion(comprising a polymer) and a fluorogenic portion (comprising one or morefluorophores), wherein one or more purine-containing residues of thepolymer are substituted with a residue comprising a pyrazolopyrimidineand/or pyrrolopyrimidine base analogue, are provided. Polymers for usein the method can comprise PNA, DNA, RNA or chimeras thereof; and thebase analogue can be present in any of the PNA, DNA or RNA portions of achimeric polymer. Any number of purine residues in the polymer can besubstituted by a base analogue. In a preferred embodiment, the method ispracticed using a composition in which a purine residue in the polymericportion that is directly adjacent to the fluorogenic portion issubstituted with a pyrazolopyrimidine or a pyrrolopyrimidine. In anotherpreferred embodiment, oligomers containing three or more consecutive Gresidues have their consecutive G residues replaced by PPG. Compositionsfor use in this method can optionally comprise a fluorescence quencherand/or a minor groove binder.

In additional embodiments, methods for detecting a target sequence in anamplification reaction, utilizing the compositions of the invention, areprovided. In a preferred embodiment, the amplification reactioncomprises a hydrolyzable probe assay.

Also provided are oligomer microarrays wherein at least one of theoligomers described herein is present on the array.

Methods for detecting a target sequence in a polynucleotide, wherein thepolynucleotide is present in a sample, by hybridization to a compositionas described herein are also provided. In a preferred embodiment, thetarget sequence has a single nucleotide mismatch with respect to arelated sequence that is also present in the sample, and the compositionforms a hybrid with the target sequence but not with the relatedsequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows real-time fluorescence analyses of a series of hydrolyzableprobe assays in which probes containing G runs of between 2 and 9nucleotides (SEQ ID NOS: 1-8) were used as fluorescent probes andcompared to probes in which the G residues were substituted by PPG (SEQID NOS:9-16).

DETAILED DESCRIPTION

The practice of the invention will employ, unless otherwise indicated,conventional techniques in organic chemistry, biochemistry,oligonucleotide synthesis and modification bioconjugate chemistry,nucleic acid hybridization, molecular biology, microbiology, genetics,recombinant DNA, and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Maniatis, Fritsch & Sambrook, MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory Press (1982); Sambrook, Fritsch &Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, ColdSpring Harbor Laboratory Press (1989); Ausubel, et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons (1987 and annualupdates); Gait (ed.), OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH,IRL Press (1984); Eckstein (ed.), OLIGONUCLEOTIDES AND ANALOGUES: APRACTICAL APPROACH, IRL Press (1991).

The disclosures of all publications and patents cited herein are herebyincorporated by reference in their entirety.

Definitions

The terms deazapurine and pyrrolopyrimidine are used interchangeably toindicate a heterocyclic nucleus comprising fused pyrimidine and pyrrolerings, according to the following general formula:

The term pyrazolopyrimidine refers to a heterocyclic nucleus comprisingfused pyrimidine and pyrazole rings, according to the following generalformula:

A “monomer” refers to a composition comprising a base or a base analoguecovalently linked to a reactive moiety, such that the monomer can beincorporated, via the reactive moiety, as part of an oligomer orpolymer. In certain cases, functional groups on the base/base analogueportion and/or on the reactive moiety are blocked so as not to bereactive during polymerization. In preferred embodiments, the reactivemoiety is an aminoethylglycine moiety, in which case the monomer can bedenoted a “PNA monomer.”

An “oligomer” is a polymer comprising linked monomer units. Oligomerscan be synthesized by sequential joining of monomers, via their reactivemoieties, as is known in the art. An oligomer can comprise a DNAoligomer, a RNA oligomer, a PNA oligomer, or any chimeric oligomer madeup of DNA, RNA, and/or PNA monomers.

A “blocking group” or “protecting group” is any chemical moiety capableof preventing reactivity of a N, S or O atom to which it is attached,under conditions in which such N, S or O atom might otherwise bereactive. Exemplary protecting groups include, but are not limited totert-butyloxycarbonyl (tBoc), 4-methoxyphenyldiphenylmethyl (MMTr),isobutyryl (iBu), 9-fluoronylmethyloxycarbonyl (Fmoc), —C₆H₅ (benzyl),diphenylcarbamoyl (DPC), 2-N-dimethylvinyl (Dmv), benzyloxycarbonyl(Cbz), benzoyl (bz), isobutanoyl, acetyl, and anisoyl (An) groups. Theseand additional protecting groups useful in the synthesis of nucleic acidand PNA oligomers are known in the art. Uhlmann et al. (1998) Angew.Chem. Int. Ed. 37:2796-2823; Green, et al. in Protective Groups inOrganic Synthesis, 2^(nd) Edition, John Wiley and Sons, Inc, NY., pp.441-452.1991.

Oligomers

The invention provides oligomers in which one or more purine bases aresubstituted with a base analogue having the same base-pairingspecificity as the purine which it replaces. The analogues can bepyrazolopyrimidines or pyrrolopyrimidines. Oligomers can comprise DNAoligonucleotides, RNA oligonucleotides, PNA oligomers, or chimerasthereof A chimera refers to an oligomer which comprises more than onetype of subunit, e.g., a RNA/DNA chimera, a PNA/DNA chimera, a RNA/PNAchimera or a PNA/DNA/RNA chimera. For chimeric oligomers, a baseanalogue can be present in any portion of the chimera (i.e., in a DNAportion, a RNA portion and/or a PNA portion).

Methods for the synthesis of DNA, RNA and PNA oligomers are known in theart. See, for example, U.S. Pat. No. 5,419,966; Gait, supra; Eckstein(ed.) “Oligonucleotides and Analogues: A Practical Approach,” 1991, IRLPress, Oxford; Ogilve et al. (1988) Proc. Natl. Acad. Sci. USA85:5746-5748; Nielsen et al. (1991) supra; Uhlmann et al. (1998) supra;U.S. Pat. Nos. 5,539,082; 5,714,331; 5,773,571; 5,736,336 and 5,766,855.Additional modified DNA and/or RNA oligomers can also be used. Forexample, methods for the synthesis of 2′-O-methyl oligoribonucleotideshave been described. Sproat et al. (1989) Nucleic Acids Res.17:3373-3386.

In general, methods for oligomer synthesis comprise stepwise cycles ofmonomer addition to a growing oligomer chain that is optionally attachedto a solid support, wherein the growing oligomer chain optionallycontains protected functional groups and a blocked growing end.Typically, at each cycle of monomer addition, the support-bound growingchain is first subjected to conditions that de-block the growing end,then condensed with a monomer, which monomer is optionally activated forcondensation. De-blocking conditions and reagents, as well as activatingconditions, and reagents, are known in the art. The monomer additionstep is repeated as often as necessary, with the identity of the monomeradded at each step corresponding with the desired sequence of theoligomer. When the desired sequence has been obtained, the nascentoligomer is subjected to conditions that deprotect functional groupsand/or cleave the completed oligomer from the support, then purified, ifnecessary.

PNA oligomers are often used as substitutes for DNA oligonucleotides invarious hybridization and other techniques. However, PNA oligomers areprone to aggregation and often exhibit reduced solubility in aqueoussolvents, especially G-rich PNAs. In a preferred embodiment, a PNAoligomer comprises one or more residues in which a purine base issubstituted by a pyrazolopyrimidine or pyrrolopyrimidine base analogue;for example, G is substituted by PPG or 7-deazaguanine, A is substitutedby PPA or 7-deazaadenine, and either G or A is substituted, by PPI or7-deazahypoxanthine. In this way the base analogue retains thebase-pairing specificity of the base for which it is substituted. In amore preferred embodiment, a PNA oligomer with one or more G residuessubstituted by PPG is provided. Such PPG-substituted PNAs exhibitreduced intermolecular and intramolecular self-association compared tooligomers containing G. This facilitates purification and handling ofthe oligomers and provides improved hybridization properties (e.g.,increased sensitivity), especially for probe sequences containing threeor more consecutive G residues.

Because a base analogue retains the base-pairing specificity of the baseit replaces, oligomers of the invention are capable of sequence-specificbinding to complementary sequences and can exhibit enhanced duplex andtriplex formation to single- and double-stranded targets, respectively.

Without wishing to be bound by any theory, applicants note that, whencompared to naturally-occurring purine bases, pyrazolopyrimidine andpyrrolopyrimidine base analogues are less likely to form non-canonicalbase pairs (such and G-T and G-G base pairs), yet retain the ability tofor canonical base pairs: characteristic of the purines which theyreplace (i.e., PPG-C, 7PG-C, PPA-T and 7PA-T base pairs).

Base Analogues and Their Synthesis

Base analogues in oligomers and in intermediates for oligomer synthesisare provided. The base analogues have a structure as indicated inFormula 1, wherein R₁ and R₂ are independently —H, —OH, —SH, or —NH₂; R₃is —H, —CN, halogen (F, Cl, Br or I), or —R₁₂—Y, wherein R₁₂ is C₁-C₁₂alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH₂ or —SH; X is ═CH— or═N—; and L is the linkage to an oligomer backbone, such as DNA, RNA, PNAor any chimera thereof.

When X is ═N—, the base analogues are pyrazolopyrimidines and when X is═CH—, the base analogues are pyrrolopyrimidines (also known as7-deazapurines). For example, when X is ═N—, R₁ is —OH, R₂ is —NH₂, andR₃ is —H, the base analogue is pyrazolopyrimidinylguanine (PPG). When Xis ═N—, R₁ is —NH₂, and R₂ and R₃ are —H, the base analogue ispyrazolopyrimidinyladenine (PPA). When X is ═N—, R₁ is —OH, and R₂ andR₃ are —H, the base analogue is pyrazolopyrimidinylhypoxanthine (PPI).

When X is ═C—, R₁ is —OH, R₂ is —NH₂, and R₃ is —H, the base analogue is7-deazaguanine (7PG). When X is ═C—, R₁ is —NH₂, and R₂ and R₃ are —H,the base analogue is 7-deazaadenine (7PA). When X is ═C—, R₁ is —OH, andR₂ and R₃ are —H, the base analogue is 7-deazahypoxanthine (7PI).

PPG and 7-deazaguanine have the same base-pairing properties as guanine(i.e., base pair with C), while PPA and 7-deazaadenine have the samebase-pairing properties as adenine (i.e., base pair with T and U). PPIand 7-deazahypoxanthine have base pairing properties equivalent to bothG and A and therefore will pair with C, T and U.

Oligonucleotides comprising the base analogues are synthesized byautomated methods that are well-known in the art, using precursors (“PNAmonomers”) according to Formula 3. The monomers are produced usingintermediates having the structure represented in Formula 2.

Allowed functional groups in Formulas 1 and 2 are as follows.

R₁ and R₂ are independently —H, —OH, —OR₆, —SH, —NH₂ or —NHR₇;

R₃ is —H, —CN, halogen (F, Cl, Br or I), or —R₁₂—Y, wherein R₁₂ isC₁-C₁₂ alkyl, alkenyl or alkynyl and Y is —H, —OH, —NH₂ or —SH;

R₄ is —H or a protecting group selected from the group consisting oftert-butyloxycarbonyl (tBoc), 4-methoxyphenyldiphenylmethyl (MMTr),isobutyryl (iBu) and 9-fluoronylmethyloxycarbonyl (Fmoc);

R₅ is —H or —C₆F₄H (TFP);

R₆ is —H, —C₆H₅ (benzyl) or a diphenylcarbamoyl (DPC) group;

R₇ is a protecting group selected from the group consisting of2-N-dimethylvinyl (Dmv), benzyloxycarbonyl (Cbz), monomethoxytrityl(MMtr), benzoyl (bz), isobutyryl (iBu), isobutanoyl, acetyl, and anisoyl(An) groups; and

X is ═CH— or ═N—.

These formulas include all isomers and tautomers of the moleculessignified thereby. Preferred embodiments of precursors for PNA synthesisand intermediates used in the synthesis of these precursors are asfollows. When R₁ is —OH, R₂ is —NH₂, R₃ is —H and X is ═N— in Formula 2,the resulting structure is2-(6-amino-4-hydroxypyrazolo[5,4-d]pyrimidinyl) acetic acid (PPGA). WhenR₁ is —NH₂, R₂ is —H, R₃ is —H and X is ═N— in Formula 2, the resultingstructure is 2-(4-aminopyrazolo[5,4-d]pyrimidinyl) acetic acid (PPAA).The corresponding derivatives of Formula 3, wherein R₄ and R₅ are —H,are abbreviated MPPGA and MPPAA, respectively. Blocked derivatives ofthese compounds are also provided, as described infra.

The designation “pyrazolo[5,4-d]pyrimidine,” as used herein, refers tothe same structures that were designated pyrazolo[3,4-d]pyrimidines inprevious co-owned publications, patents and patent applications. See,for example, U.S. Pat. No. 5,824,796; PCT WO 99/51621 and PCT WO99/51775. The reason for this change in nomenclature is so that thenames by which the structures are identified comply with those assignedto the structures by the nomenclature programs NamExpert andNomenclator, provided by ChemInnovation Software, San Diego, Calif.

The synthesis of pyrazolopyrimidine and pyrrolopyrimidine bases isaccomplished by methods known in the art. Seela et al. (1985) supra;Seela et al. (1986a), supra; Seela et. al. ((986b); supra; and Seela etal. (1987) supra. Using the reactions described by Uhlmann et al. (1998)supra for the synthesis of 2-substituted purine acetic acid derivatives,appropriately protected 4-aminopyrazolo[5,4-d]pyrimidine (PPA) and6-amino-4-hydroxypyrazolo[5,4-d]pyrimidine (PPG) can be reacted withalkyl 2-bromoacetate to give products of Formula 2. Since alkylation canoccur on both the 1 and 2 nitrogen atoms of pyrazolopyrimidines,separation of isomers and purification of the 1-substituted isomer isrequired. In the case of 7-deazapurines and related pyrrolopyrimidines,reaction with alkyl 2-bromoacetate yields only the N¹-substitutedproduct.

Accordingly, PPGA (3) can be synthesized from4-methoxypyrazolo[5,4-d]pyrimidine-6-ylamine (4) (Seela et al. (1985)Heterocycles 23:2521-2524) by alkylation with ethyl 2-chloroacetate inthe presence of sodium hydride, followed by separation of the isomers(Reaction Scheme 1).

Another approach to the synthesis of PPGA is shown in Reaction Scheme 2.In this case, 2-amino-4-6-dichloropyrimidine-5-carboxyaldehyde (1) isreacted with ethyl 2-(hydrazinol) acetic acetate to give ethyl2-(6-amino-4-{2-[(ethoxycarbonyl)methyl]hydrazino}pyrazolo[5,4-d]pyrimidinyl)acetate(2). Treatment of (2) with sodium hydroxide followed by hydrogenperoxide yields the desired product PPGA (3). An advantage of thissynthetic procedure is that it yields only the N¹-substituted isomer.See Example 1, infra.

For use in automated chemical synthesis of oligomers, reactive groups onthe base analogues, such as amino groups, must be protected. In oneembodiment, blocked derivatives of PPGA are synthesized as described inReaction Scheme 3. PPGA (3) is reacted with isobutanoyl chloride indimethylformamide and triethylamine to generate a PPGA derivative withan isobutyryl-blocked amino group (14). See Example 2, infra.

Methods for the synthesis of aminoethylglycyl derivatives of PPGA, PPAA,2-(2-amino-4-hydroxypyrrolo[2,3-d]pyrimidin-7-yl) acetic acid (7PGA) and2-(4-aminopyrolo[2,3-d]pyrimidin-7-yl)acetic acid (7PAA), for use asmonomers in automated oligomer synthesis, are known in the art. Uhlmannet al., supra. These methods involve condensation of appropriatelyprotected aminoethylglycine, e.g., methyl2-[(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)amino]acetate(MMTrAeg, Will et al. (1995) Tetrahedron 51:12069-12082) with any ofPPGA, PPAA, 7PGA or 7PAA (also protected, if necessary) in the presenceof a condensing reagent such as(O-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), orO-[(cyano(tethoxycarbonyl)methylen)amino]-1,1,3,3-tetramethyluroniumtetrafluoroborate (TOTU), as shown in Reaction Scheme 4. The R₅protecting group is chosen such that it can be removed selectively,(i.e., without removing other blocking groups) to yield a compound 7 inwhich R₅ is —H and, for example, R₁ is —NHCbz, R₂, and R₃ are —H and R₄is —MMTr. This protected derivative of MPPAA is used in the synthesis ofa PNA oligomer or a PNA/DNA chimera.

Exemplary synthesis of a blocked PPG monomer for PNA synthesis isaccomplished according to Reaction Scheme 5. PPG (15) is reacted withisobutanoyl chloride to generate an amino-blocked PPG (16), which istreated with sodium hydride and then reacted with alkyl bromoacetate togenerate, for example, a methyl acetate derivative (17). Alkalinehydrolysis of methyl ester 17 yields the acetic acid derivative 18.Further reaction of 18 with methyl2-[(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)amino] acetate(MMTrAeg) generates an alkyl ester (in this example, the methyl ester)of an MMTr-blocked aminoethylglycyl derivative with a MMTr-protectedamino group (19), which, after alkaline hydrolysis of the ester, yieldsthe MMTr-protected aminoethylglycine derivative 20. See Example 3,infra.

An exemplary method for synthesis of a PNA monomer comprising the baseanalogue PPA is shown in Reaction Scheme 6.4-aminopyrazolo[5,4-d]pyrimidine (PPA, Compound 8) is reacted with4-methoxybenzoyl chloride in pyridine to yield the amino-protected PPAderivative (9). This is reacted with sodium hydride followed by the2-bromoacetate methyl ester, and the N¹-substituted methylacetatederivative (10) is isolated. Treatment of 10 with sodium hydroxideconverts the methyl ester to the N-Bz-protected acetate derivative ofPPAA (Compound 11). See Example 4 for details.

Continuing with Reaction Scheme 6, conversion of N-Bz PPAA (11) to areactive monomer for PNA synthesis proceeds by condensation of 11 withmonomethoxytritylaminoethylaminoacetate(MMTrAeg=monomethoxytritylaminoethylglycine) to form 12, followed bytreatment of 12 with alkali to generate the MMTr-protectedaminoethylglycine derivative 13. See Example 5 for details.

A preferred PPG monomer is the 2-N-dimethylvinyl protectedMMTr-aminoethylgycine derivative (24), whose synthesis is shown inReaction Scheme 7. 4-methoxypyrazolo[5,4]pyrimidine-6-ylamine (21) wasreacted first with KOH in dry methanol, followed by reaction with methylbromoacetate to give the methyl acetate derivative (22). Alkalinehydrolysis to yield the acetic acid derivative 23 was followed byreaction with (dimethoxymethyl)dimethylamine to give (24). Reaction of(24) with MMTrAeg yielded the protected PPG monomer 25.

A preferred PPA monomer is the 2-N-dimethylvinyl protectedMMTr-aminoethylgycine derivative (29), whose synthesis is shown inReaction Scheme 8. Pyrazolo[5,4]pyrimidin-4-ylamine (8) was reactedfirst with KOH in dry methanol followed by reaction with methylbromoacetate to give the methyl acetate derivative-(26). This wasreacted with (dimethoxymethyl)dimethylamine to give (27), which wastreated with NaOH to yield (28). Reaction of (28) with MMTrAeg yieldedthe PPA monomer (29).

Synthesis of reactive derivatives of PPI follows similar procedures.Pyrazolo[5,4-d]pyrimidin-4-ol (PPI, Tominaga et al (1990) J. Heterocycl.Chem. 27:775-783) can be alkylated directly with methyl bromoacetate,followed by alkaline hydrolysis, to yield2-(4-hydroxypyrazolo[5.4-d]pyrimidinyl) acetic acid, which can beconverted as described (Uhlmann et al. (1998) supra) to the MMTr-blockedaminoethylglycine derivative. Alternatively, the hydroxyl group of PPIcould be blocked with a diphenylcarbamoyl group before reaction withmethyl bromoacetic acetate.

The same synthetic approaches used to synthesize reactive derivatives ofpyrazolo[5,4-d]pyrimidines can be used to synthesize reactivederivatives of 7-deazapurines for use in the synthesis of PNA-containingoligomers. The basic difference between the synthesis of these two typesof compounds is that in the latter case only one isomer is generatedfollowing alkylation with methyl bromoacetate.

Synthesis of PNA-containing Oligomers

In addition to the monomers and precursors described supra, theinvention includes PNA oligomers, DNA oligonucleotides and/or PNA/DNAchimeras comprising at least one monomeric unit of Formula 4, optionallycovalently attached to: one or more ligands; tail moieties or pendantgroups. A PNA oligomer comprises two or more PNA monomers that arecovalently linked by peptide bonds, as illustrated in Formula 4, where Bis a base (i.e. a heterocyclic base A, G, C, T or U as are commonlyfound in nucleic acids or a modified derivative thereof) or baseanalogue; k is between 0 and 50, preferably between 0 and 40, morepreferably between 0 and 30, and still more preferably between 0 and 20;and R₂₁ are independently —H, —OH, —NH₂, —NHR₂₂, —N(R₂₂)₂, a protectinggroup, a reactive group or an oligomer, where R₂₂ is —H or C₁₋₆ alkyl,alkenyl or alkynyl.

The synthesis of PNA oligomers from monomeric precursors is known in theart. See, for example, Uhlmann et al., supra. Synthesis is begun with aCPG resin or other solid support, containing a conjugated amino group.An appropriately blocked monomer (corresponding to the terminal monomerof the desired oligomer) is covalently coupled to the amino group withthe aid of a coupling reagent. After deprotection of the blocked growingend of the first monomer, a second monomer is coupled. The process isrepeated until an oligomer of the desired length and sequence isobtained, at which time the oligomer is cleaved from the solid supportand any base protecting groups are removed.

In one embodiment, a PNA oligomer contains a —NH₂ group at the end thatwas cleaved from the solid support; and a —COOH or —OH group at theopposite end. The terminal functional groups provide sites for theattachment of additional molecules and pendant groups to thePNA-containing oligomer.

Strategies for the synthesis of PNA/DNA chimeric oligomers are wellknown in the art. See, for example, Uhlmann et al., supra. Two principalstrategies for the synthesis of PNA/DNA chimeras are block condensationof presynthesized PNA and DNA oligomers in solution and stepwise solidphase synthesis with suitably protected PNA and DNA monomericprecursors. Those skilled in the art will appreciate that, depending onthe method of synthesis, different connecting groups between the PNA andDNA portions are possible. Exemplary linkages include, but are notlimited to, N-(2-hydroxyethyl)glycine and5′-amino-2′,5′-dideoxynucleoside phosphoramidite linkages. Uhlmann etal., supra.

Coupling reagents (or activating agents) for use in the condensation ofPNA monomers to form a PNA oligomer include, but are not limited to,benzotriazolyl-1-oxy-trispyrodinophosphonium hexafluorophosphate(PyBOP), O-(7-azabenzotriazol1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU),O-(7-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt),N,N′-diisopropylcarbodiimide (DIC), bromo tris(pyrrolidino)phosphoniumhexafluorophosphate (ByBrop), andO-[(cyano(ethoxycarbonyl)methylene)amino} 1,1,3,3-tetramethyluroniumtetrafluorborate (TOTU). These and additional activating and condensingagents are known to those of skill in the art. See, for example, Uhlmannet al., supra.

Additional molecules which can be covalently coupled to an oligomerinclude, but are not limited to, intercalators, lipophilic groups, minorgroove binders, major groove binders, reporter groups (includingfluorescent, chemiluminescent and radioactive reporters), proteins,enzymes, antibodies, chelating agents and/or cross-linking agents. Thesemolecules can be attached internally and/or at one or both ends of theoligomer. The nature and attachment of such molecules tooligonucleotides are presently well known in the art, and are described,for example, in U.S. Pat. Nos. 5,512,667 and 5,419,966 and in PCTpublication WO 96/32496, which are incorporated herein by reference.

The oligomers of the invention can also have a relatively low molecularweight tail moiety attached at either or both ends. By way of example, atail molecule can be a phosphate, a phosphate ester, an alkyl group, anaminoalkyl group, a hydrophilic group or a lipophilic group. The tailmoiety can also link an intercalator, lipophilic group, minor groovebinder, reporter group, chelating agent and/or cross-linkingfunctionality to the oligomers of the invention. The nature of tailmoieties and methods for obtaining oligonucleotides with various tailmoieties are also described in the above-referenced U.S. Pat. Nos.5,512,667 and 5,419,966.

Molecules can be attached to an oligomer of the invention to modify itssolubility in aqueous solvents. Such molecules include, but are notlimited to, saccharides and charged molecules such as amino acids,charged minor groove binders, and the like.

In a preferred embodiment, oligomers of the invention containing PPGsubstituted for guanine and/or PPA substituted for adenine also comprisea conjugated minor groove binder (MGB). Optimal single-nucleotidemismatch discrimination is obtained using MGB-conjugatedoligonucleotides containing PPG in place of guanine, as disclosed inco-owned PCT publication WO 99/51775. Polar MGBs are preferred; morepreferred MGB moieties include the trimer of3-carbamoyl-1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃)and the pentamer of N-methylpyrrole-4-carbox-2-amide (MPCS). AdditionalMGB moieties that will find use in the practice of the present inventionare disclosed in co-owned U.S. Pat. No. 5,801,155 and co-owned PCTpublication WO 99/51621, the disclosures of which are incorporatedherein by reference.

Oligomers can comprise base analogues in addition to the purineanalogues disclosed herein such as, for example, modified pyrimidinesand pyrimidine analogues.

Furthermore, the oligomers of the invention can comprise backbones otherthan a peptide backbone, or can comprise heterogeneous backbones made upof mixed peptide and non-peptide linkages. For example, oligomers withbackbones based on glycine methyl esters, ornithine, proline,diaminocyclohexane and the phosphoramidite of 2-aminopropanediol can beused. Uhlmann et al., supra. In addition, PNAs in which the peptide bondis replaced by a phosphonic acid bridge, such asN-(2-aminoethyl)phosphonoglycine and N-(2-hydroxyethyl)phosphonoglycine,can be used. Peyman et al. (1997) Angew. Chem. Intl. Ed. Engl.36:2809-2812; Efimov et al. (1998) Nucl. Acids. Res. 26:566-577.Additional oligomer linkages will be apparent to those of skill in theart.

Triplex-Forming Oligomers

PNA-containing oligomers are useful for detection of bothsingle-stranded and double-stranded nucleic-acid targets. For detectionof double-stranded nucleic acids, an oligomer binds in the major grooveof a double-stranded-target via Hoogsteen, reverse Hoogsteen orequivalent base pairing, as is known in the art. See, for example,Fresco, U.S. Pat. No. 5,422,251; Hogan, U.S. Pat. No. 5,176,996; andLampe (1997) Nucleic Acids Res. 25:4123-4131. Substitution of purines bybase analogues in a PNA-containing oligomer, as disclosed herein,facilitates triplex formation. Triplex-forming oligonucleotidesoptionally contain conjugated groups, such as fluorophores, fluorescentquenchers and any of the additional molecules described supra. In apreferred embodiment, a triplex-forming PNA-containing oligomer with oneor more purines substituted by a base analogue comprises a conjugatedminor groove binder. See supra for disclosure of minor groove bindersuseful in the oligomers of the invention.

Fluorophores and Fluorescence Quenchers

In one embodiment, an attached reporter group is a fluorescent label ora fluorophore/fluorescent quencher pair. In a preferred embodiment, thereplacement of one or more purine residues by pyrazolopyrimidine and/orpyrrolopyrimidine base analogues, in a probe containing a fluorescentlabel, results in reduced quenching of the label. Accordingly,fluorescently-labeled probes comprising one or more purine analogues,optionally comprising a fluorescence quencher, are provided.

Fluorescent labels include, but are not limited to, dyes such asfluoresceins, rhodamines, naphthylamines, coumarins, xanthenes,acridines, benzoxadiazoles, stilbenes, pyrenes, cyanines,phycoerythrins, green fluorescent proteins, and the like. Additionalfluorescent labels, and methods for their conjugation to nucleic acidand PNA probes, are known to those of skill in the art. See, forexample, Haugland (1996) Handbook of Fluorescent Probes and ResearchChemicals, Sixth edition, Molecular Probes, Inc., Eugene, Oreg. and PCTpublication WO 99/40226. In general, methods for attachment of afluorescent label and/or a fluorescence quencher to a PNA oligomer or aPNA portion of a chimeric oligomer are similar to those used forconjugating a fluorophore and/or fluorescence quencher to a DNAoligonucleotide. The fluorophore or quencher is either attached to atail moiety comprising a reactive group such as, for example, —OH or—NH₂; or is attached to a base, for example, at the 5 position of apyrimidine, the 7-position of a purine, or the 3-position of apyrazolopyrimidine or pyrrolopyrimidine.

In certain embodiments of the present invention, oligomers comprisingboth a fluorescent label (fluorophore) and a fluorescence quencher areused. A fluorescence quencher is also referred to as a quenching portionof a probe or polymer. Fluorescence quenchers include those moleculeswhose absorption spectrum overlaps the fluorescence, emission spectrumof a particular fluorophore, such that they are capable of absorbingenergy emitted by a fluorophore so as to reduce the amount offluorescence emitted (i.e., quench the emission of the fluorescentlabel). Different fluorophores are quenched by different quenchingagents. In general, the spectral properties of a particularfluorophore/quencher pair are such that one or more absorptionwavelengths of the quencher overlaps one or more of the emissionwavelengths of the fluorophore.

Appropriate fluorophore/quencher pairs, in which emission by thefluorophore is absorbed by the quencher, are known in the art. See, forexample, Haugland, supra. Exemplary pairs of fluorescencequencher/fluorophore pairs which can be used in the practice of theinvention are as follows. A preferred fluorophore/quencher pair isfluorescein and tetramethylrhodamine. Nitrothiazole blue quenchesfluorescence emission of six different dyes, namely 6-FAM, dR110, dR6G,dTMR, dROX and JAZ. Lee et al. (1999) Biotechniques 27:342-349.6-carboxytetramethylrhodamine (TAMRA) quenches emission from6-carboxyfluorescein (FAM): and 6-carboxy-4,7,2′,7′-fluorescein (TET).Lee et al. (1993) Nucl. Acid Res. 21:3671-3766.6-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino) hexanoic acid quenchesfluorescence by 7-dimethylaminocoumarin-4-acetate. Bicket et al. (1994)Ann. NY Acad. Sci. (September 6)732:351-355. 6-carboxy-X-rhodamine (ROX)and erythromycin B quench FAM emission. Li et al. (1999) Bioconj. Chem.10:241-245. The 2,4-dinitrophenyl group quenches(R,S)-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid. Hawthorne et al.(1997) Anal. Chem. 253:13-17. Dabcyl is used as a quencher of dansylsulfonamide in chemosensors and in fluorogenic peptides as a quencherfor the fluorophore EDANS. Rothman et al. (1999) Bioorg. Med. Chem.Lett. 22:509-512 and Matayoshi et al. (1990) Science 247:954-958. QSY-7is a quencher of tetramethylrhodamine. Haugland supra. Additionalfluorophore/quencher pairs can be selected by those of skill in the artby comparison of emission and absorption wavelengths according to theproperties set forth above.

Although any fluorescent label is useful in the practice of theinvention, preferred fluorophores have emission maxima between 400 and800 nm. Similarly, although any fluorescence quencher is useful,preferred fluorescence quenchers have absorption maxima between 400 and800 nm.

In a further embodiment, an oligomer comprises a pair of fluorophorescapable of fluorescence resonance energy transfer (FRET). In this case,two fluorophores are used in a FRET series. The first fluorophore(fluorescence donor) has an emission spectrum that overlaps theexcitation spectrum of the second fluorophore (fluorescence acceptor).Accordingly, irradiation at the excitation wavelengths of thefluorescence donor results in fluorescence at the emission wavelength ofthe acceptor. It is clear that any number of fluorophores, havingappropriate overlap of their emission and excitation wavelengths, canform a FRET series or three, four or more fluorophores.

In one embodiment, a fluorophore is a latent fluorophore, as disclosedin co-owned U.S. patent application Ser. No. 09/428,236, entitled“Hybridization triggered fluorescent detection of nucleic acids”, filedOct. 26, 1999.

Exemplary Advantages

When an oligomer is used as a probe or primer, substitution of baseanalogues for purines reduces aggregation of the substituted oligomer,both with itself and with other oligomer molecules. Reduction ofaggregation was demonstrated for G-rich probes as described in Example6, infra. Consequently, improved methods for detection of targetsequences by hybridization, using oligomers as probes, are obtainedusing the oligomers disclosed herein. Target sequences can comprise DNA,RNA, or any oligo- or polynucleotide.

Replacement of purines by base analogues in fluorescently-labeled probesreduces quenching of the label that occurs in unsubstituted probes. SeeExamples 7 and 8, infra. In particular, the inventors have determinedthat detection of amplification product using probes containing morethat three consecutive G residues adjacent to a fluorescent label isinefficient and, for probes containing 5 or more consecutive G residuesadjacent to a fluorescent label, no detection of product is observed.The inventors have also determined that, when PPG is substituted for G,fluorescent probes containing up to 9 consecutive PPG residues adjacentto a fluorescent label provide highly efficient detection ofamplification products. Accordingly, improved methods for detecting atarget sequence which utilize probes comprising a polymeric portion(typically an oligomer, preferably a PNA oligomer or a PNA/DNA chimera,more preferably a DNA oligomer) and a fluorescent portion are obtainedusing the compositions disclosed herein.

Thus, DNA, RNA, PNA and chimeric oligomers, comprisingpyrazolopyrimidine and pyrrolopyrimidine base analogues as disclosedherein, are useful in techniques including, but not limited to,hybridization, primer extension, hydrolyzable probe assays amplificationmethods (e.g., PCR, SSSR, NASBA), single nucleotide mismatchdiscrimination, allele-specific oligonucleotide hybridization,nucleotide sequence analysis, hybridization to oligonucleotide arrays,in situ hybridization and related techniques. Oligomers disclosed hereincan be used as immobilized oligomers in oligomer arrays such as thosedescribed in, for example, U.S. Pat. Nos. 5,492,806; 5,525,464;5,556,752 and PCT publications WO 92/10588 and WO 96/17957. Improvedspecificity and sensitivity likely result from increased solubility,decreased tendency for aggregation, reduced quenching of conjugatedfluorogenic labels, and/or some combination of these and other factors.

Improved performance of PPG-substituted probes in a real-timehydrolyzable probe assay is demonstrated in Example 9, infra.

In another embodiment of the invention, a PNA-containing oligomer withone or more purine residues substituted by a base analogue is used as apharmaceutical, for example as an antisense or anti-gene reagent, as acomponent of a ribozyme, or for gene therapy. Therapeutic uses includeD-loop formation in vivo or ex vivo.

The following examples are provided to illustrate, but not to limit, theinvention.

EXAMPLES Example 1 Synthesis of2-(6-amino-4-hydroxypyrazolo[5,4-d]pyrimidinyl)acetic acid (PPGA,Compound 3)Ethyl-2-(6-amino-4-{2-[ethoxycarbonyl)methyl]hydrazino}pyrazolo[5,4-d]pyrimidinyl)acetate(Compound 2)

2-Amino-4-6-dichloropyrimidine-5-carboxyaldehyde (Compound 1) (10 g; 52mmole) is treated with a solution of 10.1 g (64.8 mmole) of ethyl2-(hydrazinol) acetic acetate hydrochloride in 100 ml water.Triethylamine (15 ml; 107 mmole) was added and the mixture was heated to60° C. for 10 min, then stirred at room temperature for 3 days. Althoughethyl 2-(hydrazinol) acetic acetate hydrochloride did not dissolvecompletely, TLC on SiO₂ (CH₂Cl₂:CH₃OH 10:1) showed the formation of newproduct. The mixture was evaporated to dryness, taken up in toluene (100ml) and evaporated to dryness. The solid was suspended in about 300 mlCH₃CN and filtered through a SiO₂ column (49×6 cm), washed with 0.7 l ofCH₃CN and about 300 ml of CHCl₃. The filtrate was evaporated to dryness,dissolved in 120 ml hot CH₃OH and crystallized overnight at 4° C. Theproduct, a colorless solid (3.2 g) was collected and dried. TLC andreversed phased HPLC indicated a pure compound and NMR analysissupported structure 2.

2-(6-amino-4-hydroxypyrazolo[5,4-d]pyrimidinyl)acetic acid (Compound 2)

Compound 2 (3.16 g; 9.4 mmole) was dissolved in 100 ml of hot methanol,then 100 ml of a 2N NaOH solution was added, and the mixture wasrefluxed for 6 hours, at which time analysis by TLC indicated hydrolysisof the ester. Product 3 (PPGA) was formed by the addition of 2 ml of 30%H₂O₂ (in portions of 0.5 ml) to the reaction mixture, followed byheating to 80° C., until generation of O₂ from degradation of excessH₂O₂ was complete. Methanol was removed by heating at 100-120° C.,followed by cooling to room temperature and addition of 17 ml ofconcentrated HCl to give a pH of about 4. Precipitation of the productinitiated at this point, and was facilitated by the addition of ice. Theproduct was filtered, washed with cold water and dried over NaOH andP₂O₅ (yield 3.9 g). NMR confirmed the structure and indicated thepresence of about 4-8 molecules of H₂O per molecule of product.

Example 2 Synthesis of2-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]aceticacid (14)

PPGA (Compound 3, 5.58 g; 20 mmole) is suspended in anhydrous DMF (40ml) and triethylamine (4.29 ml; 30.8 mmole). Isobutanoyl chloride (2.12g; 19.9 mmole) is added dropwise using a syringe. The mixture is stirredat 100° C. for 3 hours, then treated with methanol and evaporated todryness. The residue is treated with 20 ml. 1N HCl and then withmethanol and evaporated to dryness. The residue is treated with hotisopropanol and the precipitated product is filtered off and dried invacuo. The product (14) is analyzed by TLC and HPLC and, if necessary,is purified further by chromatography.

Example 35-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]-3-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-4-oxopentanoicacid (20) N-(4-hydroxypyrazolo[5,4-d]pyrimidin-6-yl)-2-methylpropanamide(16)

Compound 15 (PPG, 3.02 g; 20 mmole) is suspended in anhydrous DMF (40ml) and triethylamine (1.45 ml, 10.4 mmole), and isobutanoyl chloride(2.12 g, 19.9 mmole) is added dropwise using a syringe. The mixture isstirred at 100° C. for 3 hours. The reaction mixture is then treatedwith methanol and evaporated ton dryness. The residue is treated withmethanol and evaporated to dryness. The residue is then treated with hotisopropanol and the precipitated product (16) is filtered off and driedin vacuo. The product is analyzed by TLC and HPLC and, if necessary, isfurther purified by chromatography.

Methyl2-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]acetate(17)

Compound 16 (4.42 g; 20 mmole) is suspended in dry DMF (40 ml), sodiumhydride (0.5 g; 20.8 mmole) is added in portions, and the mixture isstirred at room temperature for 60 min. Methyl bromoacetate (1.9 ml;20.6 mmole) is then added at room temperature, by syringe, and stirringis continued at room temperature. At completion of the reaction(monitored by TLC), the reaction mixture is treated with a small amountof carbon dioxide in methanol. The solvent is then evaporated and theresidue dissolved in CH₂Cl₂, washed once with water and then evaporatedto dryness. The product is purified by chromatography to yield thedesired isomer (17).

2-[4-Hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]aceticacid (18)

Compound 17 (4.41 g; 15 mmole) is suspended in 25 ml water, and a 2Naqueous solution of sodium hydroxide is added drop-wise at 0° C., whilemaintaining the pH at 11, until the methyl ester is completelyhydrolyzed. The reaction solution is then filtered, and the filtrate isbrought to pH 3 using 2M KHSO₄ solution, then extracted with ethylacetate. The aqueous phase is evaporated and the product (18) ispurified by chromatography.

Methyl5-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]-3-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-4-oxopentanoate(19)

Methyl 2-[(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)amino]acetate (MMTrAeg, 1.26 g; 3.1 mmole) is dissolved in DMF (8ml). To this solution is added N-ethylmorpholine (1.07 g; 6.28 mmole),3-hydroxy-4-oxo-3,4-dihdyro-1,2,3-benzotrazine (HOObt) (0.505 g; 3.1mmole), Compound 18 (0.91 g; 3.1 mmole) and diisopropylcarbodiimide(DIPC) (0.59 g; 3.72 mmole). The reaction mixture is stirred for 48hours at 4° C., at which time the solvent is evaporated and the residuedissolved in ethyl acetate. The ethyl acetate solution is washed withwater and washed once with saturated KCl solution. The organic phase isthen dried over Na₂SO₄, filtered and evaporated. The residue isdissolved in a small volume of ethyl acetate and cooled on ice to inducecrystallization of diisopropylurea, leaving the product 19 in theaqueous phase. Alternatively, the diisopropylurea is separated by silicagel chromatography from compound 19.

5-[4-hydroxy-6-(2-methylpropanoylamino)pyrazolo[5,4-d]pyrimidinyl]-3-(2{[(4-methoxyphenyl)diphenethyl]amino}ethyl)-4-oxopentanoicacid (20)

Compound 19 (1.33 g; 2 mmole) is dissolved in 10 ml dioxane. Thissolution is cooled to 0° C. and 1 M aqueous NaOH (8.66 ml) is addeddrop-wise in 5 aliquots over 2.5 hours. After an additional 2 hours atroom temperature the solution is adjusted to pH 5 by drop-wise additionof 2M KHSO₄. Precipitated salts are filtered off and washed withdioxane, and the combined filtrates are evaporated. The residue isco-evaporated with ethanol and methanol/CH₂Cl₂, then purified by silicagel chromatography to yield (20).

Example 4 Synthesis of2-{4-[4-Methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}aceticacid (Compound 11, Reaction Scheme 6)4-(Methoxyphenyl)-N-pyrazolo[4,5-d]pyrimidin-4-ylcarboxamide (9).

Pyrazolo[5;4-d]pyrimidine-4-ylamine (8) (13.5 g; 0.10 mole) is suspendedin dry pyridine (250 ml), and 4-methoxybenzolyl chloride (17.1 g; 0.1mole) is added drop-wise using a syringe. The mixture is heated at 100°C. until TLC shows that the reaction is complete (about 1 to 3 hours).The cooled reaction is then treated with methanol and the solventevaporated. The residue is co-evaporated twice with toluene and thenstirred with hot isopropanol. This mixture is cooled slowly and theprecipitated product (9) is filtered off and evaluated for purity by TLCand HPLC. If necessary, the product is further purified bychromatography.

Methyl2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}acetate(10)

Compound (9) (6.7 g; 25 mmole) is suspended in 75 ml of dry DMF. Sodiumhydride (0.65 g; 27 mmole) is added in portions, and the mixture isstirred at room temperature for 30 min. Methyl bromoacetate (2.44 ml;26.5 mmole) is added at room temperature using a syringe. Stirring iscontinued at room temperature until analysis by TLC indicates completionof the reaction, at which time the reaction mixture is treated with asmall amount of carbon dioxide in methanol. The solvent is evaporatedand the residue is dissolved in CH₂Cl₂, washed once with water and thenevaporated to dryness. The product is purified by chromatography toyield the desired isomer (10).

2-{4-[4-Methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}aceticacid (11)

Compound (10) (5.13 g; 15 mmole) is suspended in 120 ml water, and 2Naqueous sodium hydroxide solution is added drop-wise at 0° C. tomaintain the pH at 11, until the methyl ester is completely hydrolyzed.The reaction solution is filtered and the pH of the filtrate is broughtto 3, using 2M KHSO₄ solution, leading to the precipitation of product(11). The precipitate is washed with a small amount of water, dried invacuo, and analyzed for purity. If necessary, the product (11) ispurified further by chromatography.

Example 5 Synthesis of2-[N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}acetylamino]acetic acid (Compound 13, Reaction Scheme6) Methyl2-[N-(2-{[(4-methoxyphenyl)diphenylmethyl]amino}ethyl)-2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl}acetylamino]acetate(12)

Methyl 2-[(2-{[(4-methoxyphenyl)diphenylmethyl]amino} ethyl)amino]acetate (MMTrAeg, 1.26 g; 3.1 mmole) is dissolved in DMF (8 ml). To thissolution is added N-ethylmorpholine (1.07 g; 6.28 mmole),3-hydroxy-4-oxo-3,4-dihyro-1,2,3-benzotrazine (HOObt) (0.505 g; 3.1mmole), Compound 11 (1.01 g; 3.1 mmole) and diisopropylcarbodiimide(DIPC) (0.59 g; 3.72 mmole). The reaction mixture is stirred for 48hours at 4° C., then the solvent is removed in vacuo and the residue isdissolved in ethyl acetate. This solution is washed with water andwashed once with saturated KCl solution. The organic phase is dried overNa₂SO₄, filtered and evaporated. The residue is dissolved in a smallvolume of ethyl acetate and cooled in ice to induce crystallization ofdiisopropylurea, leaving the product (12) in solution. Alternatively,diisopropylurea is separated from (12) by silica gel chromatography.

2-[N-(2-{[(4-Methoxyphenyl)diphenylmethyl]amino}ethyl)-2-{4-[(4-methoxyphenyl)carbonylamino]pyrazolo[5,4-d]pyrimidinyl]acetylamino}aceticacid (13)

Compound (12) (1.43 g; 2 mmole) is dissolved in dioxane (10 ml). Thesolution is cooled to 0° C., and 1 M aqueous NaOH (8.66 ml) is addeddrop-wise in 5 aliquots over 2.5 hours. After an additional 2 hours atroom temperature, the pH is adjusted to 5 by drop-wise addition of 2MHKSO₄. Precipitated salts are filtered off, washed with dioxane, thenthe combined filtrates are dried in vacuo. The residue (13) isco-evaporated with ethanol and methanol/CH₂Cl₂, then purified by silicagel chromatography.

Example 6 Reduction in Self-Association of PPG-ContainingOligonucleotides

In this example, 13-mer and 14-mer oligonucleotide conjugates,containing between two and nine G residues, were analyzed bynondenaturing gel electrophoresis and compared with oligonucleotides ofidentical sequence except that all G residues were replaced by PPG. Thelengths and sequences of the oligonucleotides are given in Table 1.Electrophoresis was conducted in 8% polyacrylamide gels run in 1×TBEbuffer for 45 min at 40° C. Gels were stained with Daiichi 2D SilverStain II® and R_(f) values for the stained oligonucleotide bands weredetermined using two control oligonucleotides as standards. Controloligonucleotide A had the sequence 5′-ACCTGTATTCCTTGCC-3′ (SEQ ID NO.22) and control oligonucleotide B had the sequence 5′-ZTACAZCAAATZZAA-3′(SEQ ID NO. 23), where Z represents PPG.

TABLE 1 Oligonucleotide Sequences SEQ ID NO. SEQ ID NO. Sequence* Length(with G) (with PPG) 5′-CAAATGGGGGGGGG-3′ 14 1 9 5′-ACAAATGGGGGGGG-3′ 142 10 5′-AACAAATGGGGGGG-3′ 14 3 11 5′-CAACAAATGGGGGG-3′ 14 4 125′-ACAACAAATGGGGG-3′ 14 5 13 5′-CACAACAAATGGGG-3′ 14 6 145′-CACAACAAATGGG-3′ 13 7 15 5′-AGCACAACAAATGG-3′ 14 8 16 *Alloligonucleotides contained, at their 5′ ends, a conjugated fluoresceinmoiety and, conjugated at their 3′ ends, a minor groove binder (CDPI₃)and a quencher (tetramethylrhodamine). Synthesis of this type ofconjugate is described in co-owned PCT Publication WO 99/51775, thedisclosure of which is incorporated by reference.

Results of the/analysis are shown in Table 2. R_(f) values were measuredseparately for G- and PPG-containing oligonucleotides with respect tocontrol oligonucleotides A and B respectively. However, the distancemigrated by control oligonucleotides A and B was essentially identical.Oligonucleotides containing three or more G residues (oligonucleotides1-6) show a reduction in R_(f) when compared to similar-sizedoligonucleotides containing two G residues or less (e.g.,oligonucleotides 7 and 8 and control oligonucleotide A), indicatingaggregation of G-rich oligonucleotides. By contrast, oligonucleotidescontaining between two and nine PPG residues have R_(f)'s that aresimilar to one another and to a control oligonucleotide containing twoPPG residues. It was also noted that G-containing oligonucleotidesexhibited diffuse bands upon electrophoresis (the R_(f) values for theseoligonucleotides was determined by measuring from the center of theband). Furthermore, comparison of a G-containing oligonucleotide with anoligonucleotide of the same size and sequence, but with G substituted byPPG, shows that the reduced R_(f) characteristic of oligonucleotidescontaining three or more G residues is not observed with PPG-containingoligonucleotides, suggesting little or no aggregation ofoligonucleotides containing up to nine-consecutive PPG residues.

TABLE 2 Rf values of G- and PPG-containing oligonucleotides SEQ ID NO: #G # PPG R_(f) 22 2 1.00 1 9 0.58 2 8 0.42 3 7 0.37 4 6 0.35 5 5 0.32 6 40.29 7 3 0.96 8 2 0.96 23 4 1.00 9 9 0.96 10 8 0.97 11 7 0.95 12 6 0.9513 5 0.98 14 4 1.03 15 3 0.98 16 2 0.96

Example 7 Reduced Fluorescence Quenching in Fluorescently-Labelednucleotides when PPG is substituted for G

Fluorescein was coupled to GMP and to PPGMP (i.e., the monophosphatederivatives of G and PPG) and the fluorescence of 200 nM solutions ofthese conjugates was determined. Excitation was at 494 nm andfluorescence emission was measured at 522 nm. Fluorescence emission ofthe GMP conjugate was 15,447 units; while the fluorescence emission ofthe PPGMP conjugate was 32,767 units. Thus, quenching of the fluorophoreby guanine was relieved when-PPG was substituted for guanine, leading toan increase in fluorescence yield of the PPGMP conjugate of overtwo-fold, compared to the G conjugate.

Example 8 Reduced Fluorescence Quenching in Fluorescently-LabeledOligonucleotide Probes when PPG is Substituted for G

Fluorescein-oligonucleotide conjugates were examined for the effect, onfluorescence yield, of substituting PPG for G. The oligonucleotideportion of the conjugates contained a 5′-terminal G or PPG residue, towhich was coupled a fluorescein molecule. The conjugates optionallycontained a covalently coupled CDPI₃ molecule at their 3′-end. Sequencesare given in Table 3. Fluorescence of a 200 nM solution of theconjugates, in 20 mM Tris-HCl; pH 7; 40 mM NaCl; 5 mM MgCl₂, wasmeasured at room temperature, with excitation at 494 nm and emissiondetected at 0.522 nm. Results are given in Table 3.

TABLE 3 Effect fPPG substitution on fluorescence yield of ligonucleotideconjugates SEQ ID % No Sequence* F^(†) ΔF^(‡) increase 185′-Fl-GTCCTGATTTTAC-MGB-3′ 8,650 19 5′-Fl-(PPG)TCCTGATTTTAC-MGB-3′10,739 2,089 24 20 5′-Fl-GTCCTGATTTTAC-3′ 14,883 215′-Fl-(PPG)TCCTGATTTTAC-3′ 23,835 8,952 38 *Fl denotes fluorescein; MGBdenotes a conjugated minor groove binder (CDPI₃) ^(†)denotesfluorescence yield, in arbitrary units ^(‡)indicates the increase influorescence of a PPG-containing oligonucleotide, compared to aG-containing oligonucleotide

The results indicated that substitution of PPG for G increasedfluorescence (i.e., reduced quenching) by 24% and 38% for MGB-conjugatedand non-MGB-conjugated oligonucleotides, respectively.

Example 9 Improved Performance of Probes Containing Multiple ConsecutiveG Residues in a Hydrolyzable Probe Assay when PPG is Substituted for G

The oligonucleotide conjugates whose sequences are shown in Table 1 wereused as fluorescent probes in a hydrolyzable probe assay, U.S. Pat. No.5,210,015; Livak et al. (1995) PCR Meth. App. 4:357-362; Wittwer et al.(1997a) Biotechniques 22:130-138; and Wittwer et al (1997b)Biotechniques 22:176-181. The performance of G-containing probes wascompared to that of PPG-containing probes. Probes contained a conjugatedfluorophore at their 5′ end, along with a quencher and a minor groovebinder conjugated to the 3′ end of the probe, as described in co-ownedPCT publication WO 99/51775. The target sequence was5′-CACCTCAGCCTCCCAAGTAACTTTTAACCCCCCCCCATTTGTTGTGCTGTTTTCATACCTGTAATCCTGGCACTTT-3′ (SEQ ID NO. 17). Underlined portions ofthe target sequence correspond to the primer sequences. Amplificationwas conducted in an Idaho Technologies LC-24 LightCycler® with real-timefluorescence monitoring. Amplification reactions contained 10⁵ copies/μlof the target 76-mer (as above), 100 nM of each primer, 10 nMfluorescent probe (as above), 20 mM Tris-HCl, pH 7, 40 mM NaCl, 5 nMMgCl₂, 0.05% bovine serum albumin, 0.5 mM each dNTP, 0.038 Unit/μl Taqpolymerase and 0.01 Unit/μl Uracil-N-glycosylase. The cycling programwas one cycle of 50° C. for 3 min, then 95° C. for 2 min, followed by 50cycles of 95° C. for 2 sec, then 60° C. for 30 sec.

The results are shown in FIG. 1. In this method, production ofamplification product is indicated by an increase in fluorescence withtime, caused by hydrolysis of the probe hybridized to the amplificationproduct. The results obtained herein show that detection ofamplification product using probes containing more that threeconsecutive G residues was inefficient and, in fact, for probescontaining 5 or more consecutive G residues, no detection of product wasobserved. By contrast, when PPG was substituted for G in the fluorescentprobe, probes containing up to 9 consecutive PPG residues providedhighly efficient real-time detection of amplification product.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatvarious changes and modifications can be practiced without departingfrom the spirit of the invention. Therefore the foregoing descriptionsand examples should not be construed as limiting the scope of theinvention.

1. An oligonucleotide probe comprising a 5′-end, a 3′-end, one or moredetectable fluorescent labels, and a qenching agent which qenches thefluorescence emission of the one or more fluorescent labels wherein theprobe comprises at least 4 consecutive guanine residues in which atleast one of the four consecutive guanine residues has been replaced bya PPG residue, the probe exhibiting reduced quenching of the one or morefluorescent labels due to said replacement of residues.
 2. Anoligonucleotide probe of claim 1, further comprising an attached minorgroove binder.
 3. An oligonucleotide probe of claim 1, wherein the labelis a fluorescein.
 4. An oligonucleotide probe of claim 1, wherein thelabel is a cyanine.
 5. An oligonucleotide probe of claim 1, wherein thelabel is a rhodamine.
 6. An oligonucleotide probe of claim 2, whereinthe minor groove binder is located at the oligonucleotide 5′ end.
 7. Anoligonucleotide probe of claim 2, wherein the minor groove binder islocated at the oligonucleotide 3′ end.
 8. An oligonucleotide probe ofclaim 1, wherein the label is located at the oligonucleotide 5′ end. 9.An oligonucleotide probe of claim 1, wherein the label is located at theoligonucleotide 3′ end.
 10. An oligonucleotide probe of claim 2, whereinthe minor groove binder is selected from the group consisting of atrimer of 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI₃)and a pentamer of N-methylpyrrole-4-carbox-2-amide (MPC₅).
 11. Anoligonucleotide probe of claim 2, further comprising multiplefluorescent labels.
 12. An oligonucleotide probe of claim 11, whereinthe emission wavelengths of one of the fluorescent labels overlaps theabsorption wavelengths of another of the fluorescent labels.
 13. Anoligomeric probe of claim 1, comprising from 4 to 9 consecutive guanineresidues in which at least one of the guanine residues has been replacedby a PPG residue.
 14. An oligomeric probe of claim 1, comprising atleast 4 consecutive guanine residues in which all of the at least fourconsecutive guanine residues have been replaced by PPG residues.
 15. Anoligomeric probe of claim 14 comprising from 4 to 9 consecutive guanineresidues in which all of the guanine residues have been replaced by PPGresidues.
 16. An oligonucleotide probe of claim 1, wherein at least oneguanine radical that is directly adjacent to a fluorescent label hasbeen replaced by a PPG residue.
 17. An oligonucleotide probe of claim16, comprising from 4 to 9 consecutive guanine radicals directlyadjacent to a fluorescent label in which at least one of the guanineresidues has been replaced by a PPG residue.
 18. An oligomeric probe ofclaim 17 comprising from 4 to 9 consecutive guanine residues in whichall of the guanine residues have been replaced by PPG residues.